Abstract
Vacuolar H+-pumping pyrophosphatases (VPs) provide a proton gradient for Na+ sequestration in the tonoplast; however, the regulatory mechanisms of VPs in developing salt tolerance have not been fully elucidated. Here, we cloned a barley (Hordeum vulgare) VP gene (HVP10) that was identified previously as the HvNax3 gene. Homology analysis showed VP10 in plants had conserved structure and sequence and likely originated from the ancestors of the Ceramiales order of Rhodophyta (Cyanidioschyzon merolae). HVP10 was mainly expressed in roots and upregulated in response to salt stress. After salt treatment for 3 weeks, HVP10 knockdown (RNA interference) and knockout (CRISPR/Cas9 gene editing) barley plants showed greatly inhibited growth and higher shoot Na+ concentration, Na+ transportation rate and xylem Na+ loading relative to wild-type (WT) plants. Reverse transcription quantitative polymerase chain reaction and microelectronic Ion Flux Estimation results indicated that HVP10 likely modulates Na+ sequestration into the root vacuole by acting synergistically with Na+/H+ antiporters (HvNHX1 and HvNHX4) to enhance H+ efflux and K+ maintenance in roots. Moreover, transgenic rice (Oryza sativa) lines overexpressing HVP10 also showed higher salt tolerance than the WT at both seedling and adult stages with less Na+ translocation to shoots and higher grain yields under salt stress. This study reveals the molecular mechanism of HVP10 underlying salt tolerance and highlights its potential in improving crop salt tolerance.